The present invention relates to a method for amplifying nucleic acid, a method for assaying DNA fragments in mixture, a method for assaying RNA fragments in mixture, and an expression profiling process of a group of expressed genes; more specifically, the invention relates to a method for assaying DNA fragments by gel electrophoresis and by using fluorophore label.
The nucleotide sequence of a gene or a DNA can be compared with other nucleotide sequences by DNA sequencing, but it is very difficult to determine the sequence of a long DNA or a mixture sample of a great number of DNA fragments. So as to determine the sequence of such long DNA, therefore, the long DNA is first fragmented; then, the resulting DNA fragments are assayed on gel electrophoresis patterns. Due to the recent progress of various assay methods and apparatuses therefor, numerous expressed genes can simultaneously be detected and assayed.
The expression profiling method includes for example a scanning process based on gel electrophoresis. The scanning process includes FDD [Fluorescent Differential Display; FEBS Letters 351, 231-236 (1994)] and a process using terminal nucleotide selective primer [Nucleic Acids Research, 24, 2616-2617 (1996); Nucleic Acids Symposium Series, No. 35, 257-258 (1996)].
All these scanning processes comprise PCR (Polymerase Chain Reaction) using plural primers in common to plural DNA fragments along with a sample cDNA or mRNA as template and recovering gene expression information based on the electrophoresis patterns of the resulting PCR products. Because the expression can be detected with no use of any gene-specific probe according to the scanning processes, advantageously, the expression profile of a gene with a nucleotide sequence not yet identified can be yielded.
The process using terminal nucleotide selective primer comprises digesting a double-stranded cDNA with a restriction enzyme and ligating an oligonucleotide with a known nucleotide sequence at the terminus of a DNA fragment as a digestion product. PolyA tail is generally present at the 3' terminus of cDNA. Because a nucleotide sequence between the polyA tail at the 3' terminus of the cDNA and the ligated oligonucleotide is specific to the cDNA, a DNA fragment carrying the nucleotide sequence is assayed as a fragment representing (or identifying) the cDNA.
Because gene species of several thousands to several ten thousands in number are expressed in living organisms, the types of such cDNA representative fragments are so numerous that these representative fragments cannot be separated and assayed in one lane by electrophoresis. Thus, all these types of such representative fragments are divided in plural groups, whereby the number of such DNA fragments included in each one group is sufficiently reduced such that the DNA fragments in each one group can be separated and assayed satisfactorily by gel electrophoresis. So as to divide all the representative fragments in plural groups, PCR primers with a selective nucleotide sequence composed of two nucleotides at the 3' terminus thereof are used. By PCR, DNA fragments with terminal two nucleotides complementary to each selective nucleotide sequence are amplified. From two primer sets with two selective nucleotide sequences is selected each one primer; subsequently, a group of a combination of appropriate two primers is prepared.
By PCR with primers of the individual sets, the resulting amplified products are assayed by electrophoresis. Via combinations of DNA fragments expressed on the electrophoresis pattern recovered by using the primers of the individual sets, the whole information of each expressed gene can be recovered. Consequently, the type of each expressed gene and the expression level thereof can be identified.
For fragment assay, at least one primer of oligo dT primer and a primer complementary to the oligomer ligated at the terminal digestion site of a DNA fragment is labeled with fluorophore; using the primer labeled with the fluorophore, the DNA fragment is amplified in such a number above the detection sensitivity of an assay apparatus such as fluorescent DNA sequencer and the like.